Dendrimer biocide-silver nanocomposites: their preparation and applications as potent antimicrobials

ABSTRACT

A novel cationic dendrimer biocide-silver nanocomposite and methods for its use as a biocide. The biocidal nanocomposites of the present invention are effective against a variety of microbial species, including anthrax. The invention is also highly stable and safe for exposure to human skin. The invention has applications as an antibiological warfare agents, antimicrobial agent for surface coatings and as a general biocide that is safe for human exposure.

CLAIM OF PRIORITY

[0001] Priority is claimed under 35 U.S.C. §119(e) from the U.S.Provisional Application Serial No. 60/210,888 filed Jun. 9, 2000; whichis herein incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to the synthesis andcharacterization of a series of new compositions of matter,formulations, and applications of dendrimer biocide-silvernanocomposites as potent antimicrobial agents.

[0004] 2. Description of the Related Art

[0005] Dendrimers are well defined, highly branched macromolecules thatemanate from a central core. Commercially available dendrimers includepolyamidoamine (“PAMAM”) dendrimers and polypropylene imine (“PPI”)dendrimers. Dendriditic architecture brings a very high number offunctional groups in a compact space. Dendrimers in the presentinvention can for example be selected from the group consisting ofpolyamidoamine dendrimers, polylysine based dendrimers, polyethyleneoxide based dendrimers, silicon based dendrimers, polyether baseddendrimer and polypropylene imine dendrimers. A polylysine baseddendrimers refers to a dendrimer in which the backbone or structureconsists essentially of polylysine. A polyethylene oxide baseddendrimers refers to a dendrimer in which the backbone or structureconsists essentially of polyethylene oxide. A silicon based dendrimersrefers to a dendrimer in which the backbone or structure consistsessentially of silicon. A polyether based dendrimer refers to adendrimer in which the backbone or structure consists essentially ofpolyether.

[0006] The advent of dendrimers represents a major breakthrough insynthetic chemistry. Dendrimers can be tailored to generate uniform ordiscrete functionalities and possess tunable inner cavities, surfacemoieties, sizes, molecular weights, and solvent interactions. Dendrimerscan be synthesized by a convergent approach, see Tomalia, et al.,Macromolecules, 20 at 1164 (1987), alternatively, dendrimers can also besynthesized by a divergent approach, see Tang, et al., BioconjugateChem., 7 at 703-714 (1996).

[0007] In the divergent approach, growth of dendrimers starts from amulti-functional core. Through a series of reaction and purificationsteps, dendrimers grow radially outwards. At different stages of thesynthesis, dendrimers are identified by generations. As the generationincreases, the number of functional groups, the size of the dendrimer,and the molecular weight of the dendrimer increase. Commerciallyavailable dendrimers, such as polyamidoamine (PAMAM) dendrimers fromDendritech Inc. (Midland, Mich., USA) and polypropylene imine (PPI)dendrimers from DSM (Geleen, Netherlands) are synthesized by thedivergent approach.

[0008] In the convergent approach, dendrons, as parts of dendrimers, aresynthesized according to the divergent approach and these dendrons arethen coupled to a multifunctional core. The advantage of the convergentapproach is that the chemistry of each dendron can be different, anddistinct functional groups can be integrated into dendrimers at precisesites. Due to the repetitive nature of the dendrimer synthesis and theextensive purification required, dendrimers are very expensive and notreadily available. The combination of discrete numbers offunctionalities in one molecule and high local densities of activegroups has attracted a lot of attention, especially for biologicalapplications. The unique architecture of dendrimers, they have beeninvestigated for a wide variety of applications, such as gene deliveryvesicles, Tang, et al., Bioconjugate Chem., 7 at 703-714 (1996);Kukowska-Latallo, et al., Proc. Natl. Acad. Sci USA, 93 at 4897-4902(1996), catalysts, Zeng, F. Z., S. C. Chem. Rev., 97 at 1681 (1997);Newkome, et al., Chem. Rev., 99 at 1689-1746 (1999), drug deliverycarriers, Liu, M.; Frechet, J. M., J. Proc. Am. Chem. Soc. Polym. Mater.Sci. Engr., 80 at 167 (1999); Uhrich, K., TRIP, 5 at 388-393 (1997);Liu, H.; Uhrich, K. Proc, Am. Chem. Soc. Div. Polym. Chem., 38 at 1226(1997), chromatography stationary phases, Matthews, et al., Prog. Polym.Sci., 23 at 1-56 (1998), boron neutron capture therapy agents, Newkome,et al., Dendritic Macromolecules: Concepts, Syntheses, Perspectives;VCH: Weinheim, Germany (1996); Newkome, G. R., Advances in Den driticMacromolecules; JAI Press: Greenwich, Conn., Vol. 2 (1995), and magneticresonance imaging contrast agents. Tomalia, D. A. Adv. Mater., 6 at529-539 (1994).

[0009] The dendriditic architecture of dendrimers provides a very highnumber of functional groups in a compact space. Because of thisproperty, it is reasonable to expect that these novel molecules willplay a major role in materials whose performance depends on high localconcentration, such as drugs or antimicrobial agents.

[0010] The versatile chemistry of the dendrimers can also include metalatoms. The metal can be either an integral part of the dendrimer, suchas in the building block, core, or terminal group, or it can associatewith the dendrimer through interactions with branching units. Thesemetals can be metal cations, metal salts, metal oxides or even elementalmetal. Newkome and coworkers published a recent comprehensive review ondendrimers with metals (metallodendrimers) G. R. Newkome, E. He, C. N.Moorefield, Chem. Rev. 1999, 99, 1689. Metal salts, such as silver, areknown antimicrobial agents. Dendrimer nanocomposites, formed bydendrimers and antimicrobial salts, offer a new way to deliver orenhance the antimicrobial properties of these agents.

[0011] Balogh et al. synthesized dendrimer nanocomposites, dendrimerswith inorganic silver or silver ions, and tested their antibacterialproperties. Balogh, L. Proc. Am. Chem. Soc. Div. Colloi & Surf. Chem.,54. (1999). For these dendrimer nanocomposites, the dendrimer itselfdoes not have any antibacterial property. The activity comes from thesilver/silver ions. In contrast, the quaternary ammonium functionalizeddendrimers of the current invention derive antibacterial properties fromthe dendrimer itself. The dendrimers of the current invention aredifferent from all previous investigations in that the surface groups ofthe dendrimers were transformed into quaternary ammonium groups. Unlikeknown QACs, the quaternary ammonium functionalized dendrimers of thecurrent invention are more effective against Gram-negative bacteria suchas E. coli and Gram-positive bacteria such as S.aures.

SUMMARY OF THE INVENTION

[0012] The inventors have successfully synthesized novel dendrimerbiocides. These dendrimer biocides are fully described in U.S. patentapplication Ser. No. 09/588,585, which is herein incorporated byreference for all purposes. The present invention is directed to noveldendrimer biocide-silver complexes, which are new nanocompositessynthesized from the dendrimer biocides of the U.S. patent applicationSer. No. 09/588,585 and silver salts. The hybrid structures embodyingthe present invention provide even more potent antimicrobial properties.

[0013] The structure investigated in this study is not very clear. Atentative name of “dendrimer silver nanocomposite” was used. The potentbiocide properties of the dendrimer-silver nanocomposites according tothe invention all came by surprise to the inventors.

[0014] In the copending patent application Ser. No. 09/588,585, thedescribed dendrimer biocides are capable of killing anthrax spores.However, the inventors have unexpectedly found that the dendrimerbiocide-silver nanocomposites have superior biocide properties. Thesynergy between the dendrimer biocide and the silver ion has greatpotential for denaturing spores. Because spores are a significant classof biological weapons, the present invention would be useful incombating biological warfare weapons.

[0015] Traditional antibiological warfare agents are known to be veryreactive and extremely toxic. Such agents include chlorine,formaldehyde, and peroxygen. In contrast, the novel dendrimerbiocide-silver nanocomposites of the present invention are nonreactiveand are virtually nontoxic to human skin. Similar compounds have beenused in hand-wash formulations. Thus, the invention may provide aneffective antibiological warfare agent. Examples of uses for the presentinvention include a denaturing spray or soldier uniforms impregnatedwith the invention.

[0016] Additionally, the invention can also be used in any situationthat requires a potent biocide that is also environmentally stable.These applications include incorporation into protective coatings orpaints, personal products such as cosmetics, industrial products,hospital products, and sanitation of swimming pools and spas. Theinvention can also be immobilized onto the surface to create efficientantimicrobial surfaces for use as biomaterials, antifouling paints, andother similar devices.

[0017] The object of the invention is to provide a dendrimerbiocide-silver complex, and the method of using the dendrimerbiocide-silver complex as an antimicrobial agent.

[0018] In accordance with the invention, silver nitrate (AgNO₃) is addedto a dendrimer biocide solution to obtain a novel dendrimer-silvercomplex containing both dendrimer and silver ions.

DESCRIPTION OF FIGURES

[0019]FIG. 1. Plot showing the antibacterial activity ofdendrimer-silver nanocomposites compared to dendrimer biocide alone.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The novel dendrimer biocide-silver composite compositions of thepresent invention are prepared by reacting a dendrimer biocide withsilver compound. The dendrimer biocides of the present invention arefully described in U.S. patent application Ser. No. 09/588,585, which isherein incorporated by reference. The dendrimer biocides of the presentinvention are the quatemary ammonium functionalized dendrimers ofFormula I:

[0021] where D is a dendrimer; n is the generation number of thefunctionalized dendrimer; z is an integer less than or equal to 2(n+1);X⁻ is an anion; R is a linking group; Y is an alkyl group or aryl group;A is an alkyl group or aryl group, and B is an alkyl group or arylgroup. The quaternary ammonium functionalized dendrimers are thenreacted with silver compound to form the dendrimer biocide-silvernanocomposites of the present invention.

[0022] To simplify the naming of the dendrimer biocides used in thepresent invention, a shorthand of “DnXNY” based on Formula 1 is used. Inthis shorthand format, D is a poly(propylene imine) (“PPI”) dendrimer ofgeneration n and having an anion X and a Y group. For example, “D3ClNC8”denotes a dimethyloctyl (C₈) ammonium (N) chloride (Cl) functionalizedPPI generation 3 dendrimer (D3).

[0023] The dendrimer biocide-silver nanocomposites are prepared by aprocess comprising the following steps:

[0024] (1) adding excess AgNO₃ to the dendrimer biocide solution toconvert the chloride ion of the dendrimer biocide to a nitrate ion;

[0025] (2) removing any precipitated AgCl;

[0026] (3) removing any remaining AgNO₃ in the solution by adding excessNaCl to the supernatant liquid; and

[0027] (4) diafilter to remove remaining NaCl.

[0028] In a preferred embodiment greater than 1 percent stoichiometricamount of a silver compound may be used to prepare the dendrimerbiocide-silver nanocomposite.

[0029] In the above process, the inventors unexpectedly found that therewas still some silver ions left in the solution even when excess NaClwas added in step 3. These silver ions seemed to prefer to stayassociated with the dendrimer rather than forming AgCl precipitate.

[0030] The inventors further discovered that the dendrimerbiocide-silver nanocomposite structures are only stable in water. If thewater was evaporated under vacuum, redissolution was impossible and adark powder of Ag₂O was observed with powdered dendrimer biocideresidue.

[0031] Because of these observations, the inventors believe that thenovel dendrimer biocide-silver nanocomposite compounds contain bothdendrimer and silver ions. While not wishing to be bound to a particulartheory, the inventors believe that the silver ions may be absorbed on orcomplexed with the dendrimer. Depending on the composition of thesolution, these nanocomposites can be stable for more than 3 monthscompared to several-hour stability of other dendrimer-nanocompositepreparations.

EXAMPLES

[0032] Preparation

[0033] Dendrimer biocide-silver nanocomposites were prepared byinitially reacting silver nitrate (AgNO₃) with a D3ClNC8 dendrimerbiocide to prepare a dendrimer biocide with nitrate counter-anion. Thedendrimer biocide with nitrate counter-anion was prepared according tothe method described above as follows:

[0034] 1. Adding excess AgNO₃ to a D3ClNC8 solution to convert thechloride anion of the dendrimer to nitrate ion;

[0035] 2. Precipitated AgCl was then centrifuged and excess NaClsolution was added to the supernatant liquid to remove any remainingAgNO₃ in the solution;

[0036] 3. After a dialysis to remove the remaining NaCl, a dendrimerbiocide with NO₃—counter-anion is obtained.

[0037] Preparation of Ag-200

[0038] The sample Ag-200 refers to the sample when a 200 percentstoichiometric amount of AgNO₃ was used in preparing the dendrimerbiocide-silver nanocomposite. Approximately 0.1 gram of D3ClNC16 isdissolved in minimal amount of ethanol and dilute with water to obtain10 mL solution. 10 mL of 0.1N AgNO3 is then added to change the counteranion of the dendrimer to nitrate. The solution is then centrifuged at3000 g for 2 hours. The supematant is collected and 10 mL of 0.1N NaClis added to remove the residual amount of AgNO3 in the solution. Theexcess NaCl was then removed with diafiltration of distilled water (1000MWCO membrane). The final volume after diafiltration can be adjustedaccording to the desired concentration. Typically a 100 mL solution isachieved.

[0039] Preparation of Ag-500

[0040] The sample Ag-500 refers to the sample when a 500 percentstoichiometric amount of AgNO₃ was used in preparing the dendrimerbiocide-silver nanocomposite. Approximately 0.1 gram of D3ClNC16 isdissolved in minimal amount of ethanol and dilute with water to obtain10 mL solution. 25 mL of 0.1N AgNO3 is then added to change the counteranion of the dendrimer to nitrate. The solution is then centrifuged at3000 g for 2 hours. The supernatant is collected and 25 mL of 0.1N NaClis added to remove the residual amount of AgNO3 in the solution. Theexcess NaCl was then removed with diafiltration of distilled water (1000MWCO membrane).

[0041] The final volume after diafiltration can be adjusted according tothe desired concentration.

[0042] Typically a 100 mL solution is achieved.

[0043] Measurement of Antimicrobial Properties

[0044] The antimicrobial properties of these composites were evaluatedusing a bioluminescence method. For these bioluminescence experiments,several strains of recombinant E. coli are used. The recombinant E. colistrains containing a fusion of Escherichia coli heat shock promoters anda lux gene of Vibrio fischeri were developed at DuPont. Relevantinformation regarding various strains is known to those skilled in theart.

[0045]E. coli stock solutions, kept at −80° C. freezer until requiredfor use, were prepared in Lucia-Barton (LB) media supplemented with 20%glycerol. Glycerol was added to protect bacteria during freezing andthawing processes. During inoculation, a required amount of E. colisuspension (typically 10%) was added to fresh and sterile LB broth andincubated overnight at 37° C. These cells were also centrifuged andwashed twice with phosphate buffered saline. Cell concentration wasdetermined using a Petroff-Hausser counting chamber (ScientificProducts, Edison, N.J.). Bacteria suspensions were then diluted to thetest concentration. The recombinant E. coli strains were also stocked ina similar way to the wild-type E. coli. The medium used was Lucia-Barton(LB) supplemented with 25 mg/ml kanamycin monosulfate to maintain theplasmid. These plasmid-containing strains were grown to an earlyexponential phase at 30° C. The temperature used was lower than normalbecause enzymes responsible for generating light would be deactivated at37° C. These cells were also centrifuged and washed twice with 0.1%peptone water before use.

[0046] There are a variety of methods that can be used to evaluate theantimicrobial properties of new materials. Method selection oftendepends on specific applications. Antimicrobial test results might bereported qualitatively, using terms as sensitive, intermediate, orresistant, or quantitatively in terms of concentration of an agentneeded to inhibit or kill bacteria. Suspension tests are typically usedto evaluate water-soluble antimicrobials, while surface antimicrobialtests are designed to characterize the antimicrobial properties ofnon-leaching biocidal materials.

[0047] During suspension tests, the effectiveness of biocides wasquantified by mixing a suspension of viable bacteria and a certainconcentration of test materials and monitoring the subsequent number oflive cells at distinct time points. Bacteria were grown overnight andharvested as described. The plate-count method and bioluminescence testmethods are two ways to quantify live cells.

[0048] In using the plate-count method, cells were re-suspended in a PBSbuffer at a concentration between 1×108 and 1×109 cells/ml in sterile,15-ml polypropylene centrifuge tubes (VWR Scientific Products, WestChester, Pa.). The viable cell concentration was determined by making aseries of 10-fold dilutions of the cell suspension and spreading thebacteria on agar plates. The plates were then incubated over night at30° C. for recombinant E. coli and 37° C. for wild-type E. coli. Thevisible colonies on the plates were then counted in 24-48 hours. Eachcolony represents one viable bacterium from the original suspension. Thenumber of colonies, multiplied by the dilution, provides a measure ofthe viable cells in the original suspension.

[0049] In these studies, a dendrimer biocide was added to a cellsuspension. The suspension was then agitated thoroughly with a vortexer(Scientific Products, McGraw Park, Ill.). Aliquots of the suspensionwere removed after 15, 30, and 60 minutes of biocide exposure. Thealiquots were immediately used in a series of 10-fold dilutions withbuffer containing 10% Tween 80 (Aldrich Chemical Company, Milwaukee,Wis.), which was utilized as a deactivating agent for the quaternizedmoieties. Without the denaturing agents, test samples would continue toexert its toxic influence, thereby increasing the apparent activity ofthe biocide.

[0050] For the bioluminescence experiments, several strains ofrecombinant E. coli were used. These recombinant E. coli strainscontaining a fusion of Escherichia coli heat shock promoters. Wheneverbacteria receive stress from a toxic compound, the intensity of lightemitted from the bacteria will change. From a “light-on” or “light-off”response one can obtain real-time cell viability data. Strain TV 1048,in which the lux operon is coupled to the promoter of lac operon, wasused in this study. Bioluminescence is observed under normal growthconditions. Whenever the bacteria are in a biocidal environment, thelight-off response quantitatively corresponds to the antibacterialeffect.

[0051] During experiments, these plasmid-containing strains were grownto an early exponential phase at 30° C. in LB medium and then incubatedwith a known amount of the test sample. Bioluminescence was measuredeither in real time or at some time intervals using a luminometer (Model20e, Turner Design, CA) and the data were recorded by a computer withdata collection capacity.

[0052] The antibacterial property of the D3ClNC8 dendrimerbiocide-silver nanocomposite against Gram-negative E. coli wasdetermined by a bioluminescence method. Bioluminescence is observedunder normal growth conditions for the recombinant E. coli strain TV1048. Whenever the bacteria are in a biocidal environment, the light-offresponse corresponds to the toxic effect of the biocide. The result isexpressed as the sample bioluminescence normalized to a control (withoutbiocidal dendrimer of the present invention) versus time. The reductionof luminescence quantitatively shows the antibacterial activity of thesample.

[0053] At 4 ppm, the dendrimer biocide inhibited the growth of E. coli,but the bacteria could adjust to the environmental stress and survive.At higher concentrations (20 ppm), the bioluminescence decreased veryrapidly and went down to undetectable levels in 15 min indicating astrong biocidal effect. In a control experiment, the bioluminescence ofthe bacteria did not change much (5%) if the same concentration of purePPI generation 3 dendrimers was added.

[0054] The sample Ag-200 refers to the sample when a 200 percentstoichiometric amount of AgNO₃ was used in preparing the dendrimerbiocide-silver nanocomposite. The Ag-200 formulation was observed to bemuch more potent than the dendrimer biocide alone. See FIG. 1. Thisfigure shows that 2 ppm of D3ClNC8 reduces the bioluminescence to only10 percent of the bioluminescence of the control. However, formulationAg-500, containing only 0.8 ppm of D3ClNC8 and less than 0.8 ppm of Ag⁺can reduce the bioluminescence to 0.1 percent of the control. The exactsilver concentration was unknown since some AgCl precipitate consumedAg⁺). The more potent formulation, Ag-200 with 0.8 ppm of D3ClNC8 andless than 0.3 ppm of Ag⁺ can reduce the bioluminescence to 0.001 percentof the control, which corresponded to a 5-order of magnitude reduction.The inventors further observed that the Ag-200 formulation isunexpectedly more potent than Ag-500 formulation.

[0055] The improvement of the potency may be explained by synergybetween the dendrimer biocide and silver ions. Silver ions have beenused as antimicrobials for 4000 years. The mode of action of silver ionis similar to Hg²⁺. Both ions can complex with electron donor groupscontaining sulfur, oxygen or nitrogen. Silver parallels mercuric ions inrelative affinities for various proteins, but is somewhat less tightlybound. Synergism is defined as the ability of two antimicrobials actingtogether to markedly increase the rate of the bacteriocidal action ascompared to the rate of each antimicrobial alone. A typical example ofsynergy is between EDTA and QACs. EDTA is able to chelate the calciumand magnesium, thus destabilizes the phospholipid membrane and promotesthe membrane-disrupting action of QACs.

[0056] While the invention has been particularly shown and describedwith reference to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made without departing from the spirit and scope of the invention asdefined in the appended claims.

What is claimed is:
 1. A dendrimer biocide-silver nanocompositecomposition comprising a quaternary ammonium dendrimer biocide andsilver ions associated with said dendrimer biocide.
 2. The dendrimerbiocide-silver nanocomposite composition of claim 1 wherein greater than1 percent stoichiometric amount of a silver compound is used to preparethe dendrimer biocide-silver nanocomposite.
 3. The dendrimerbiocide-silver nanocomposite composition of claim 1 wherein from about200 to 500 percent stoichiometric amount of a silver compound is used toprepare the dendrimer biocide-silver nanocomposite.
 4. The dendrimerbiocide-silver nanocomposite composition of claim 1 wherein a 200percent stoichiometric amount of a silver compound is used to preparethe dendrimer biocide-silver nanocomposite.
 5. The dendrimerbiocide-silver nanocomposite composition of claim 1 wherein a 500percent stoichiometric amount of a silver compound is used to preparethe dendrimer biocide-silver nanocomposite.
 6. The dendrimerbiocide-silver nanocomposite composition of claim 1 wherein thedendrimer biocide is a D3ClNC8 dendrimer biocide.
 7. A dendrimerbiocide-silver nanocomposite prepared by a process comprising: (a)adding excess AgNO₃ to a dendrimer biocide solution to convert thechloride ion of the dendrimer biocide to a nitrate ion; (b) removing anyprecipitated AgCl; (c) removing any remaining AgNO₃ in the solution byadding excess NaCl to the supernatant liquid; and (d) diafiltering toremove remaining NaCl.
 8. A method of controlling the growth of amicroorganism comprising exposing said microorganism to a dendrimerbiocide-silver nanocomposite composition comprising a quaternaryammonium dendrimer biocide and silver ions associated with saiddendrimer biocide.
 9. A method of controlling the growth of amicroorganisms according to claim 8 wherein said microorganism isselected from the group consisting essentially of acidogenicgram-positive cocci, Gram-negative anaerobic oral bacteria, Group Astreptococci, enteric bacteria, Gram-negative rods, and Gram-positivecocci.
 10. A method of controlling the growth of a microorganismsaccording to claim 8 wherein said microorganism is selected from thegroup consisting essentially of streptococcus, staphylococci,haemophilus influenzae, escherichia coli, P. aeruginosa, burkholderiacepacia, Pseudomonas pseudomallei, C. albicansm, staphylococcusepidermidis, and S. aureus.
 11. A method of controlling the growth of amicroorganism according to claim 8 wherein said microorganism is aspore.
 12. A method of controlling the growth of a microorganismaccording to claim 11 wherein said spore corresponds to B. anthracis oranthrax.
 13. A method of controlling the growth of a microorganismaccording to claim 8 wherein said dendrimer biocide-silver nanocompositeis immobilized on a surface.
 14. A method of controlling the growth of amicroorganism according to claim 13 wherein said surface is comprised ofa polymer, or glass, or metal.
 15. A method of controlling the growth ofa microorganism according to claim 14 wherein said surface polymer isselected from the group consisting of polyurethane, polystyrene,polyethylene, and polypropylene.
 16. A method of controlling the growthof a microorganism according to claim 8 wherein said dendrimerbiocide-silver nanocomposite is applied as a spray.
 17. A method ofcontrolling the growth of a microorganism according to claim 8 whereinsaid dendrimer biocide-silver nanocomposite is incorporated into acoating.